The lysosome plays a critical role in the degradative metabolism of the cell. Its importance to the organism is evidenced by the existence of over 35 human lysosomal storage diseases. The transport of newly synthesized soluble lysosomal enzymes to lysosomes is dependent upon the mannose 6-phosphate receptors (MPRs). Phosphomannosyl residues on lysosomal enzymes-serve as high affinity ligands for binding to MPRs which results in the segregation and delivery of lysosomal enzymes to lysosomes. Two distinct, but homologous, MPRs have been identified. The 270-kDa insulin-like growth factor II/cation-independent (IGF-II/CI) MPR is a multifunctional glycoprotein that binds both mannose 6-phosphate (Man-6-P) on lysosomal enzymes and IGF-II, a nonglycosylated polypeptide hormone. The second receptor, the cation-dependent (CD) MPR, is a 46-kDa glycoprotein that exhibits optimal phosphomannosyl binding in the presence of divalent cations but, unlike the IGFII/CI-MPR, does not bind IGF-II. The long-term objectives of this proposal are: 1) to characterize the ligand binding sites of both MPRs at the molecular level to better understand the mechanism by which these receptors bind lysosomal enzymes with high affinity to target them to the lysosome and, 2) to determine the mechanism by which a single protein (IGF-II/CI-MPR) can bind two very different ligands, a carbohydrate moiety (Man-6-P) and a protein determinant (IGF-II), with high affinity. To achieve these goals, oligonucleotide-directed site-specific mutagenesis will be employed to introduce specific amino acid changes and/or deletions into the receptors to determine the location of the Man-6-P and IGF-II binding sites and to analyze the roles asparagine-linked oligosaccharides, specific amino acids and disulfide bonds have in Man-6-P and IGF-II binding. The resulting altered receptors will be expressed in Xenopus laevis oocytes and assayed for their ability to bind ligand. These altered receptors will also be expressed in either an E. coli or baculovirus expression system to allow for isolation of significant amounts of protein for quantitative binding studies. The disulfide bonding arrangement of the extracytoplasmic domains of the receptors encompassing their ligand binding sites will be determined to provide information on their tertiary structure and to aid in the x-ray crystallographic and nuclear magnetic resonance (NMR) spectroscopy analyses. In addition, soluble fragments of both receptors' ligand binding sites will be produced in milligram amounts in a suitable expression system to determine their three-dimensional structure by both X-ray crystallographic and NMR spectroscopy studies.